We report a protein immobilized self-assembled monolayer (SAM) of gold nanoparticles (GNPs) on indium-tin-oxide ( ITO) coated glass plate. The protein-antibody, Mb-Ab, was covalently immobilized over the self-assembly of GNPs through a mixed SAM of 11-mercapto undecanoic acid (MUA) and 3-mercapto propionic acid (MPA) via carbodiimide coupling reaction using N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide (EDC) and N-hydroxy succinimide (NHS). The whole assembly was constructed on 0.25 cm(2) area of ITO-glass plate (Mb-Ab/MUA-MPA/GNPs/APTES/ITO-glass) and an impedimetric study was carried out for its application in myoglobin detection. This prototype assembly was characterized by scanning electron microscopy, atomic force microscopy and electrochemical techniques. The modified electrode showed an increased electron-transfer resistance on coupling with protein antigen. Mb-Ag, in the presence of a redox probe [Fe(CN)(6)](3-/4-). Its exhibits an electrochemical impedance response to protein myoglobin-antigen, Mb-Ag, concentration in a linear range from 0.01 mu g to 1.65 mu g mL(-1) with a lowest detection limit of 1.4 ng mL(-1). (C) 2011 Elsevier B.V. All rights reserved.